Microbial Genetics

1. İ. ÇağatayAcunerM.D., Clinical Microbiologist, AssociateProfessor Department of MicrobiologyFaculty of Medicine, Yeditepe University Microbial Genetics

2. Learning Objectives • Explainbasics of nucleic acid function in bacteria • (replication of DNA, chromosome replication, DNA repair, gene expression, gene organization) • Describe sources of mutation and variation in bacteria • Describe basic features of bacteriophages and plasmids • Describe mechanisms of gene transfer in bacteria • Explain the importance of genomic plasticity in bacteria

3. Referencesand Recommended Further Readings • Medical Microbiology, 7th Ed. Murray P.R., Rosenthal K.S., Pfaller M.A. 2013, Mosby. • Mim’s Medical Microbiology, 5th Ed. Goering R.V., et al. 2012, Mosby. • Sherris Medical Microbiology, 5th Ed. Ryan K.J., et al. 2010, McGraw Hill.

4. Bacterial Genome • Bacterial genome is the total collection of genes carried by a bacterium • on chromosome • on extrachromosomal genetic elements • Genes are sequences of nucleotides that have a biologic function • Examples; • protein-structural genes (cistrons, which are coding genes), • ribosomal ribonucleic acid (RNA) genes, • recognition and binding sites for other molecules (promoters and operators) • Each genome contains many operons, which are made up of genes • Eukaryotes have two distinct copies of each chromosome;diploid • Bacteria have only one copy of their chromosomes;haploid • Because bacteria have only one chromosome, alteration of a gene (mutation) will have a more obvious effecton the cell • The structure of the bacterial chromosome is maintained by polyamines; • spermine and spermidine (not by histones) • Bacteria may also contain extrachromosomal genetic elements; • plasmids • bacteriophages(bacterial viruses) • these elements are independent of the bacterial chromosome • in most cases can be transmitted from one cell to another

5. Escherichia coli genome

6. Comparative genomes (genomics) of Escherichia coli strains

7. Transcription • Transcription; • The information carried in the genetic memory of the DNA is transcribed into a useful messenger RNA (mRNA) for subsequent translation into protein • RNA synthesis is performed by a DNA-dependent RNA polymerase • sigma factor; • recognizes a particular sequence of nucleotides in the DNA (the promoter) • binds tightly to this site • promoter sequences occur just before the start of the DNA that actually encodes a protein • sigma factors bind to these promoters to provide a docking site for the RNA polymerase • Some bacteria encode several sigma factors to allow transcription of a group of genes under special conditions, such as heat shock, starvation, special nitrogen metabolism, or sporulation • polymerase binds to the appropriate site on the DNA • sequential addition of ribonucleotides occur complementary to the sequence in the DNA • entire gene or group of genes (operon) is transcribed • RNA polymerase dissociates from the DNA • Other RNA types that are also transcribed from the DNA; • transfer RNA (tRNA) • which is used in protein synthesis • ribosomal RNA (rRNA) • a component of the ribosomes

8. DNA codon table

9. Transcription • Transcription ; • Promoters and operators control the expression of a geneby; • influencing which sequences will be transcribed into messenger RNA (mRNA) • Operons; • groups of; • one or more structural genes; • expressed from a particular promoter • ending at a transcriptional terminator • Thus all the genes • coding for the enzymes of a particular pathway; • can be coordinately regulated • Operons with many structural genes; • polycistronic • Example; • E. coli lac operon; • includes all the genes necessary for lactose metabolism, and • the control mechanisms; • turning off (in the presence of glucose) or • turning on (in the presence of galactose or an inducer) • lac operon includes; • a repressor sequence, • a promoter sequence, and • structural genes for the β-galactosidase enzyme • a permease, and • an acetylase

10. E. colilacoperon

11. Translation • Translation; • Translation is the process by which the language of the genetic code, • in the form of mRNA, • is converted (translated) into a sequence of amino acids; • the protein product

12. Translation • Translation ; • Codon; • a set of three nucleotides • each correspondtoeither; • an amino acid or • a stop or start information • 64 different codon combinations • encoding; • 20 amino acids • start (5’UTR →fMet≈AUG in prokaryotes)and termination-stop- (UAA, UGA, UAG) codons • ‘Degeneracy’ of the genetic code ; • some of the amino acids are encoded by more than one triplet codon • this feature protects the cell from the effects of minor mutations in the DNA or mRNA • Anticodon; • each tRNA molecule; • contains a three-nucleotide sequence complementary to one of the codon sequences • allows base pairing • binds to the codon sequence on the mRNA • Amino acid; • attached to the opposite end of the tRNA • corresponds to the particular codon-anticodon pair

13. Control of Gene Expression • Bacteria have developed; • mechanisms to adapt quickly and efficiently to; • changes and triggers from the environment • adaptation mechanisms allow them to; • coordinate and regulate the expression of genes • for ; • multicomponent structures or • the enzymes of one or more metabolic pathways • many bacterial genes are controlled at; • multiple levels and • by multiple methods • a coordinated change in the expression of many genesoccurs through use of a different sigma factor for the RNA polymerase(e.g. sporulation) • this would change the specificity of the RNA polymerase and allow mRNA synthesis for the necessary genes while ignoring unnecessary genes • bacteria might produce more than six different sigma factors to; • provide global regulation in response to; • stress, • shock, • starvation, • coordinate production of complicated structures(e.g.flagella)

14. Control of Gene Expression • Simple triggerscan turn on or turn off the transcription of a single gene or a group of genes; • temperature, • osmolarity, • pH, • nutrient availability, or • the concentration of specific small molecules, • such as oxygen or iron • The expression of the components of virulence mechanisms; • also coordinately regulated from an operon • Salmonellainvasion genes within a pathogenicity island are turned on by high osmolarity and low oxygen, conditions present in the gastrointestinal tract • E. coli senses its exit from the gut of a host by; • a drop in temperature, • and inactivates its adherence genes • Low iron levels; • can activate expression of hemolysin in E. coli or • diphtheria toxin from Corynebacteriumdiphtheriae, potentially to kill cells and provide iron • Quorum sensing for; • virulence factors of S. aureus • biofilm production by Pseudomonas spp.

15. Replication of DNA • Replication of the bacterial genome ; • linked to the growth rate of the cell • initiated at a specific sequence in the chromosome called OriC • requires many enzymes; • Helicase; • to unwind the DNA at the origin to expose the DNA, • Primase; • to synthesize primers to start the process, and • DNA-dependent DNA polymerase/s; • that synthesize a copy of the DNA, • but only if there is a primer sequence to add to and only in the 5' to 3' direction • new DNA is synthesizedsemiconservatively; • using both strands of the parental DNA as templates • new DNA synthesis; • occurs at growing forks • proceeds bidirectionally • leading strand is copied continuously in the 5' to 3' direction • lagging strand must be synthesized as many pieces of DNA using RNA primers (Okazaki fragments)

16. Replication of DNA • lagging-strand DNA must be extended in the 5' to 3' direction as its template becomes available • then the pieces are ligated together by the enzyme DNA ligase • to maintain the high degree of accuracy required for replication, • the DNA polymerases possess "proofreading" functions, • which allow the enzyme to confirm that the appropriate nucleotide was inserted and to correct any errors that were made • during log-phase growth in rich medium, many initiations of chromosomal replication may occur before cell division • this process produces a series of nested bubbles of new daughter chromosomes, each with its pair of growth forks of new DNA synthesis • the polymerase moves down the DNA strand, incorporating the appropriate (complementary) nucleotide at each position • replication is complete when; • the two replication forks meet 180 degrees from the origin • the process of DNA replication puts great torsional strain on the chromosomal circle of DNA; • this strain is relieved by topoisomerases (e.g., gyrase), which supercoil the DNA

17. Bacterial DNA replication • New DNA synthesis occurs at growing forks and proceeds bidirectionally. • DNA synthesis progresses in the 5' to 3' direction continuously (leading strand) or in pieces (lagging strand). • Assuming it takes 40 minutes to complete one round of replication, and assuming new initiation every 20 minutes, initiation of DNA synthesis precedes cell division. • Multiple growing forks may be initiated in a cell before complete septum formation and cell division. • The daughter cells are "born pregnant."

18. Bacterial cell division • Replication requires extension of the cell wall and replication of the chromosome and septum formation. • Membrane attachment of the DNA pulls each daughter strand into a new cell.

19. Mechanisms of Genetic Transfer between Cells • The exchange of genetic material between bacterial cells may occur by; • (1) conjugation; • which is the mating or quasisexual exchange of genetic information from one bacterium (the donor) to another bacterium (the recipient) • (2) transformation; • which results in acquisition of new genetic markers by the incorporation of exogenous or foreign DNA from the environment • (3) transduction; • which is the transfer of genetic information from one bacterium to another by a bacteriophage • (4?) Once inside a cell, a transposon can jump between different DNA molecules (e.g., plasmid to plasmid or plasmid to chromosome)

20. Transformation • Bacteria take up fragments of naked DNA and incorporate them into their genomes • First mechanism of genetic transfer to be discovered in bacteria • (1928, Griffith, +15 years Avery, MacLeod, and McCarty); • observed that pneumococcus virulence was related to the presence of a surrounding polysaccharide capsule • and that extracts of encapsulated bacteria producing smooth colonies could transmit this trait to nonencapsulated bacteria, normally appearing with rough edges • Gram-positive and gram-negative bacteria can take up and stably maintain exogenous DNA • Certain species are naturally capable of taking up exogenous DNA;competent; • Haemophilus influenzae, Streptococcus pneumoniae, Bacillus species, and Neisseria species • Most bacteria do not exhibit a natural ability for DNA uptake

21. Conjugation • Conjugation; • one-way transfer of DNA from a donor (or male) cell to a recipient (or female) cell through the sex pilus(oradhesin) • the mating type (sex) of the cell depends on; • the presence (male) or absence (female) of aconjugative plasmid; • such as the F plasmidof E. coli • F plasmid is defined as conjugativebecause; • carries all the genes necessary for its own transfer • including the ability to make sex pili • initiate DNA synthesis at the transfer origin (OriT) of the plasmid • transfers itself • converting recipients into F+ male cells • usually occurs between members of the same or related species, • but also mayoccur between prokaryotes and cells from plants, animals, and fungi • Examples; • E. coli, bacteroides, enterococci, streptococci, streptomycetes, and clostridia • Large conjugative plasmids specify; • colicin • antibiotic resistance(byadhesins)

22. Conjugation • Conjugation; • Hfr (high frequency recombination) cell; • If a fragment of chromosomal DNAhas been incorporated into the plasmid; • it is designated an F prime (F') plasmid • when it transfers into the recipient cell, • it carries that fragment with it and converts it into an F' male • if the F’plasmid sequence (F’) is integrated into the bacterial chromosome, • the cell is designated an Hfr (high frequency recombination) cell • DNA that is transferred by conjugation is a single-stranded molecule • recircularized and its complementary strand synthesized in therecipientcell • Integration of an F plasmid into the bacterial chromosome generates an Hfr cell • Conjugation results in transfer of; • a part of the plasmid sequence, and • some portion of the bacterial chromosomal DNA • because of the fragile connection between the mating pairs, the transfer is usually aborted before being completed; • such that only the chromosomal sequences adjacent to the integrated F are transferred

23. Transduction • Genetic transfer by transduction; • is mediated by; • bacterial viruses (bacteriophages); • pick up fragments of DNA • package them into bacteriophage particles • DNA is; • delivered to infected cells • becomes incorporated into the bacterial genomes • Transduction can be classified as; • specialized; • if the phages in question transfer particular genes (usually those adjacent to their integration sites in the genome) • generalized; • if the selection of the sequences is random because of accidental packaging of host DNA into the phage capsid

24. Underlying common mechanism: Recombination • Incorporation of extrachromosomal (foreign) DNA into the chromosomeoccurs by recombination. • There are two types of recombination: • homologous • nonhomologous • Homologous (legitimate) recombination; • occurs between closely related DNA sequences • generally substitutes one sequence for another • requires a set of enzymes produced (in E. coli) by the rec genes • Nonhomologous (illegitimate) recombination; • occurs between dissimilar DNA sequences • generally produces ; • insertions or • deletions or • both • requires specialized (sometimes site-specific) recombination enzymes, • such as those produced by; • many transposons • lysogenic bacteriophages

25. Plasmid • The pBR322 plasmid is one of the plasmids used for cloning DNA. • This plasmid encodes resistance to ampicillin (Amp) and tetracycline (Tet) and an origin of replication (ori). • The multiple cloning site in the pGEM plasmid provides different restriction enzyme cleavage sites for insertion of DNA within the β-galactosidase gene (lacZ). • The insert is flanked by bacteriophage promoters to allow directional messenger RNA expression of the cloned sequence.

26. Transposon • A, The insertion sequences code only for a transposase (tnp) and possess inverted repeats (15 to 40 base pairs) at each end. • B, The composite transposons contain a central region coding for antibiotic resistances or toxins flanked by two insertion sequences (IS), which can be either directly repeated or reversed. • C, Tn3, a member of the TnA transposon family. The central region encodes three genes-a transposase (tnpA), a resolvase (tnpR), and a β-lactamase-conferring resistance to ampicillin. A resolution site (Res site) is used during the replicative transposition process. This central region is flanked on both ends by direct repeats of 38 base pairs. • D, Phage-associated transposon is exemplified by the bacteriophage mu.

27. Mechanisms of bacterial gene transfer